This project aims to develop new radiolabeled substrates for the enzyme monoamine oxidase-B (MAO- B) in the human brain. In the brain, MAO-B is conspicuously expressed in astrocytes, and increased MAO-B enzymatic activity has been used as a biomarker of gliosis in numerous studies of diseases such as refractory epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, and other neurodegenerative conditions. As reactive gliosis is a recognized pathological marker of localized cellular responses to a wide variety of insults to the brain, non-invasive measurement of the extent and pattern of gliosis could have wide applicability to the localization of injured or diseased tissue (e.g., for resection in epilepsy), differentiation of related diseases (Alzheimer's versus frontal-temporal dementia (FTD) or Lewy Body disease), and evaluation of new therapeutic treatments, and thus could significantly assist in the diagnosis and management of many CNS diseases. This project proposes the design, chemical and radiochemical synthesis, and evaluation in vitro and in vivo of new carbon-11 and fluorine-18 labeled substrates for Positron Emission Tomography (PET) imaging of MAO-B enzymatic activity. In an innovative approach to MAO-B imaging, the project will prepare novel radiolabeled derivatives of 4-thiophenyl- and 4-carbamoyl-N-methyl-1,2,3,6-dihydropyridines that are known to be non-toxic MAO-B substrates that form polar reaction products. 4-Thiophenyl-1,2,3,6-dihydropyridines are oxidized to the corresponding non-toxic dihydropyridinium salts; the 4-carbamoyl-1,2,3,6- dihydropyridines are oxidized to the corresponding dihydropyridinium salts that spontaneously hydrolyze to release polar amines. The polar radiolabeled products from MAO-B enzymatic oxidation will accumulate at the site of enzyme action, thus using the concept of metabolic trapping to provide quantifiable PET imaging data. Candidate radiotracers will be evaluated for in vitro reactivity with MAO- B (Km and kcat) and good in vivo brain uptake and retention in rodents using ex vivo dissection techniques. Promising radiotracers will then be evaluated for brain uptake, irreversible trapping, and overall pharmacokinetics in rhesus monkey brain by microPET imaging. The overall goal of the project is to identify appropriate radiolabeled substrates that can be further evaluated and validated for applications in human PET imaging.